Figure 4

Functional analysis of iNOS⁺ring subset. A. CFSE-labeled Clone 4 transgenic CD8⁺ T-cells stimulated with HA-peptide pulsed DC co-cultured in the presence or absence of sorted ring cells from 4T1 tumors. Selective iNOS inhibitor L-NIL was added to some wells. T cell proliferation was measured by CFSE dilution assay using flow cytometry. Data are plotted as percent CD8⁺ T-cells proliferation and are from one of three independent experiments. Error bars show SD. B. Effect of selective iNOS inhibitors L-NIL and 1,3-PB-ITU on tumor growth. Mice were treated with L-NIL or 1,3-PB-ITU via daily i.p injections starting one day after 4T-1 injection. Tumor volume was determined daily using the formula a² x b/2, where a and b are the shortest and longest perpendicular dimensions of the tumor, respectively. * The differences between the L-NIL or 1,3-PB-ITU-treated group and control (4T-1 only) were significant (p <0.0004 on days 6–15). Error bars = SD, n = 5–7 mice per group. Representative of 2 independent experiments. C&D. Intratumoral Ring and CD8⁺ T-cells numbers were quantified by flow cytometric analysis of day 9 tumors. Graphs represent the mean (±SD) of at least 5 mice from 2 independent experiments. D *The differences between the L-NIL- treated and control groups was significant (P ≤ 0.05), the differences between the 1,3-PB-ITU-treated group and control was not (p <0.1). E. The ability of Ring cells to kill CD8⁺ T-cells was evaluated in an in vitro co-culture assay. Ring cells were sorted and added at different ratios with CD8⁺ T-cells and apoptosis of CD8⁺ T-cells was measured via staining with Annexin V. The apoptosis effect was related to ring cell abundance. The gray histogram represents CD8⁺ T-cells alone versus thin open histogram (1:1 ratio of CD8⁺ T/Ring) and thick open histogram (1:2 ratio)Representative of 3 independent experiments.